Adeno-associated virus type 2 (AAV) is a non-pathogenic human parvovirus which is being developed as a gene therapy vector. The Rep78 and Rep68 proteins encoded by AAV are DNA-binding proteins involved in AAV replication, AAV gene regulation, and the preferential integration of the AAV genome into a region within the q arm of human chromosome 19. These proteins also inhibit cell proliferation and the replication of human immunodeficiency virus type 1 (HIV). Many of these effects appear to be mediated by interactions between the Rep proteins and specific binding sites within the AAV genome, the chromosome 19 integration locus, the promoters of several human proto-oncogenes and the HIV long terminal repeat promoter (HIV-LTR). A computerized search of human DNA sequence databases, using known Rep recognition sequences as query sequences revealed several potential Rep78/68 binding sites, including sites in or near the c-sis proto-oncogene and genes encoding alpha-A-crystallin, carcinoma marker GA733-1, and a hepatocyte glucose transporter. These binding sites were confirmed by electrophoretic mobility shift assays using Rep proteins expressed from the HIV-LTR in human 293 cell transient trans- fections or as maltose binding protein fusions in Escherichia coli. Since we have previously demonstrated that Rep binding sites can be involved in Rep-mediated gene regulation, we have begun to test for differential expression of these genes in the presence or absence of Rep proteins. We detected increased levels of c-sis RNA, which encodes platelet-derived growth factor B (PDGF-B), in human 293 cells transfected with a Rep68 expression plasmid. Further characterization of these Rep binding sites may allow us to understand better the effects of Rep proteins on human cells and may also lead to the identification of alternate Rep-dependent AAV integration sites within the human genome.